CN104818244A - Amnion epithelial cell separation and culture method - Google Patents
Amnion epithelial cell separation and culture method Download PDFInfo
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Abstract
The invention relates to an amnion epithelial cell separation and culture method which aims to solve the problem of low purity of the amnion epithelial cells which are separated and cultured by the existing method. The method comprises the following steps: 1. amnion tissue separation and disinfection; 2. amnion epithelial cell separation; and 3. amnion epithelial cell purification culture to obtain the high-purity P0-generation amnion epithelial cell unicell suspension. The amnion patch prepared from the amnion tissues can ensure the flatness of the amnion epithelial surface; meanwhile, the amnion back mucus is closely attached to the connective tissue and nitrocellulose membrane to enhance the trypsinization efficiency and promote the separation between the cells and tissues, so that all the amnion epithelial cells can be collected by short-time digestion, thereby avoiding the reduction of the cell wall-attachment rate; and after the short-time culture, trypsinization is performed twice to obtain the high-purity amnion epithelial cells. The method is applicable to the fields of fundamental research and clinical transplantation.
Description
Technical field
The present invention relates to a kind of method that amniotic epithelial cells is separated, cultivates.
Background technology
The amnion tissue discarded perinatal period is one of important regenerative medicine seed cell source, its wide material sources, is easy to obtain and without ethics dispute, is study hotspot in recent years.In amnion separable go out amniotic epithelial cells (human amnioticepithelial cell, and amnion mesenchymal stem cell (human amniotic mesenchymal stem cell hAEC), hAMSC), two kinds of stem cells all have rich content, immunogenicity low, be the main stem cell resource of future clinical treatment without features such as tumorigenicity, wherein amniotic epithelial cells has more " dryness ", more close to the character of embryonic stem cell, therefore become regenerative medicine area research focus.
Amniotic epithelial cells is grown by ectoderm at after fertilization on the 8th day, is lining in amnion internal surface, and major part is simple cuboidal and mast cell, participates in amniotic fluid and is formed and transmit, also can synthesize, secrete and be formed basilar membrane extracellular matrix composition.Research display, amniotic epithelial cells has certain triploblastica differentiation potential equally, express the multiple surface marker relevant to embryonic stem cell: SSEA3/4, TRA1-60/81, Nanog, Oct4, Sox2 etc., can can be divided into liver cell, cardiac-like muscle cell, neurocyte, skeletonization and chondrocyte under specific inductive condition in vitro, and immunogenicity is low, there is anti-inflammatory action.Amniotic epithelial cells also secretes the cytokine relevant with injury repairing of physiological level, as platelet-derived growth factor, vascular endothelial growth factor, angiogenesis factor, transforming grouth factor beta 2, TIMP1 and tissue inhibitor of metalloproteinase 2.These cytokines can promote kind of a cell proliferation, reduce inflammatory reaction, regulate many committed steps of injury repairing.Preclinical study display human amnion membrane has remarkable effect in the disease models such as treatment nervous tissue degenerative disease, myocardial tissue damage, diabetes, proves to have important medical value at tissue repair field amniotic epithelial cells.
The separation method of current conventional amniotic epithelial cells adopts 0.25% pancreatin one or many digestion method, because the trysinization efficiency ratio amniotic epithelial cells of amnion mesenchymal stem cell is higher, often pollutes amniotic epithelial cells, causes purity to decline.Simultaneously amnion tissue is very thin, and soft, be adhered, not easy to operate, be contained in centrifuge tube or aseptic bottle and carry out trysinization and usually fold, be deposited in together, be also difficult to after cell dissociation break away from amnion tissue, cause the excessive retention of cell in amnion tissue.The amnion back side has the materials such as mucus, bloodstain and unnecessary reticular tissue can affect trysinization efficiency simultaneously, reduce the output of amniotic epithelial cells, but the excessive prolongation of digestion time (the total digestion time of repeatedly trypsin digestion commonplace is at present at about 30-50min) can cause again, and adherence rate declines, proliferation and differentiation ability reduces, and surface antigen markers is lost.The digestion of pancreatin substep is adopted in patent CN201410050051 (Human plactnta Subaerial blue green algae and stem cell bank construction process thereof) and CN201310650384 (a kind of method producing stem cell from people's amnion), want to reach the object improving amniotic epithelial cells purity, but the fact shows to digest rear amnion tissue and often piles up to be suspended in solution and stick, wrap a large amount of amniotic epithelial cells, therefore need repeatedly repeatedly to rinse with physiological saline or D-Hanl ' s balance liquid, cause a large amount of spillings of amnion mesenchymal stem cell like this, reduce amniotic epithelial cells purity.CN201410050051 (Human plactnta Subaerial blue green algae and stem cell bank construction process thereof) adopts slide glass to remove amnion back side blood vessel, reticular tissue after amnion is peeled off, the effect of rolling directly can destroy amniotic epithelial cells, and long-time operation causes amniotic epithelial cells activity to reduce.
Summary of the invention
The present invention will solve the problem that existing method is separated, the purity of the amniotic epithelial cells of cultivation is low, provides a kind of method that amniotic epithelial cells is separated, cultivates.
The method that a kind of amniotic epithelial cells of the present invention is separated, cultivates, completes according to the following steps:
One, amnion tissue is separated and sterilization: peeled off on placenta by amnion, then use normal saline flushing 2 ~ 3 times, then soaks 3 ~ 10min in containing the physiological saline of 1% ~ 3% cefoperazone;
Two, amniotic epithelial cells is separated: amnion is cut into 50 ~ 150cm
2bulk, then the epithelial surface of the amnion after cutting is sticked upward on aseptic nitrocellulose membrane, in culture dish A, adds 3 ~ 10mL physiological saline, then the epithelial surface of amnion is posted down in culture dish A, leave standstill 2 ~ 3min, obtain amniotic membrane patch, amniotic membrane patch is placed in culture dish B, add pancreas enzyme-EDTA digestion 15 ~ 20min that 10 ~ 30ml mass concentration is 0.25%, then after sealed membrane sealing, culture dish is put into constant-temperature table to digest, obtain Digestive system, add 2 ~ 5ml foetal calf serum again and stop enzyme reaction, then remove in amniotic membrane patch and reunite, the amniotic epithelial cells be adhered, amniotic membrane patch being transferred to is equipped with in the culture dish C of physiological saline again, clean adherent amniotic epithelial cells, obtain scavenging solution, collect Digestive system and scavenging solution, through the sieved filter of 100 order cell, obtain amniotic epithelial cells single cell suspension,
Three, amniotic epithelial cells purifying is cultivated: amniotic epithelial cells single cell suspension step 2 obtained is at 4 DEG C, the pelleted by centrifugation 5 ~ 15min of 1500rpm, abandon supernatant, then the low sugar DMEM/F12 substratum re-suspended cell containing Urogastron is added, by 0.9 ~ 1.1 × 10 after mixing
5/ cm
2inoculum size be inoculated in culturing bottle, then be placed in 37 DEG C, CO
2volumetric concentration is cultivate in the incubator of 5%, after cell confluency reaches 85%, reject non-adherent cell, and with normal saline flushing 2 times, then the pancreas enzyme-EDTA digestion that mass concentration is 0.05% is added, 37 DEG C of digestion 2 ~ 5min, reject Digestive system, use normal saline flushing again 1 ~ 2 time, then the pancreas enzyme-EDTA that mass concentration is 0.25% is added, 3 ~ 6min is digested under 37 DEG C of conditions, add FBS again and stop enzyme reaction, collect Digestive system, by Digestive system at 4 DEG C, centrifugal 5 ~ 15min under the condition of 1500rpm rotation concussion, abandon supernatant, then DMEM/F12 substratum re-suspended cell is added, obtain high purity P0 for amniotic epithelial cells single cell suspension, namely complete.
Beneficial effect of the present invention has:
(1) can ensure that amnioic epithelium face is smooth after amnion tissue makes amniotic membrane patch, simultaneously amnion back side mucus and reticular tissue and nitrocellulose membrane fit tightly, promote being separated of trysinization efficiency and cell and tissue, short period of time digestion can collect whole amniotic epithelial cells, avoids the reduction of adherence rate.
(2) amniotic epithelial cells is attached to outside amnion surface substrate film, can preferentially and separate tissue, the amnion back side and nitrocellulose membrane are fitted and are hindered the outflow of amnion tissue interstitial cells, in operation, amnion tissue does not need significantly to stir amniotic epithelial cells also can be made fully to be separated with amnion tissue, reduce further the pollution of amnion mesenchymal stem cell.
(3) short period of time cultivates rear use 0.05% pancreatin, 37 DEG C of digestion 2-5min can make amnion mesenchymal stem cell be separated completely, and now amniotic epithelial cells still keeps open and flat adhered state, after purifying, add 0.25% pancreatin, 37 DEG C of digestion 3-6min can obtain high purity amniotic epithelial cells.
Accompanying drawing explanation
Fig. 1 is the microscope figure of p1 for amniotic epithelial cells form of test 1 preparation;
Fig. 2 is the microscope figure of P1 for amnion mesenchymal stem cell form of test 1 preparation;
Fig. 3 is the microscope figure of test 1 control group p1 for amniotic epithelial cells form;
Fig. 4 is that the p1 of test 1 preparation is for Cytokeratin19 immunofluorescence expression figure (× 100) in amniotic epithelial cells;
Fig. 5 is that the p1 of test 1 preparation is for DAPI immunofluorescence expression figure (× 100) in amniotic epithelial cells;
Fig. 6 is that p1 prepared by test 1 expresses integration map (× 100) for amniotic epithelial cells Cytokeratin19 and DAPI immunofluorescence;
Fig. 7 is that the p1 of test 1 preparation is for Cytokeratin19 immunofluorescence expression figure (× 200) in amniotic epithelial cells;
Fig. 8 is that the p1 of test 1 preparation is for DAPI immunofluorescence expression figure (× 200) in amniotic epithelial cells;
Fig. 9 is that p1 prepared by test 1 expresses integration map (× 200) for amniotic epithelial cells Cytokeratin19 and DAPI immunofluorescence.
Embodiment
Embodiment one: the method that a kind of amniotic epithelial cells of present embodiment is separated, cultivates, completes according to the following steps:
One, amnion tissue is separated and sterilization: peeled off on placenta by amnion, then use normal saline flushing 2 ~ 3 times, then soaks 3 ~ 10min in containing the physiological saline of 1% ~ 3% cefoperazone;
Two, amniotic epithelial cells is separated: amnion is cut into 50 ~ 150cm
2bulk, then the epithelial surface of the amnion after cutting is sticked upward on aseptic nitrocellulose membrane, in culture dish A, adds 3 ~ 10mL physiological saline, then the epithelial surface of amnion is posted down in culture dish A, leave standstill 2 ~ 3min, obtain amniotic membrane patch, amniotic membrane patch is placed in culture dish B, add pancreas enzyme-EDTA digestion 15 ~ 20min that 10 ~ 30ml mass concentration is 0.25%, then after sealed membrane sealing, culture dish is put into constant-temperature table to digest, obtain Digestive system, add 2 ~ 5ml foetal calf serum again and stop enzyme reaction, then remove in amniotic membrane patch and reunite, the amniotic epithelial cells be adhered, amniotic membrane patch being transferred to is equipped with in the culture dish C of physiological saline again, clean adherent amniotic epithelial cells, obtain scavenging solution, collect Digestive system and scavenging solution, through the sieved filter of 100 order cell, obtain amniotic epithelial cells single cell suspension,
Three, amniotic epithelial cells purifying is cultivated: amniotic epithelial cells single cell suspension step 2 obtained is at 4 DEG C, the pelleted by centrifugation 5 ~ 15min of 1500rpm, abandon supernatant, then the low sugar DMEM/F12 substratum re-suspended cell containing Urogastron is added, by 0.9 ~ 1.1 × 10 after mixing
5/ cm
2inoculum size be inoculated in culturing bottle, then be placed in 37 DEG C, CO
2volumetric concentration is cultivate in the incubator of 5%, after cell confluency reaches 85%, reject non-adherent cell, and with normal saline flushing 2 times, then the pancreas enzyme-EDTA digestion that mass concentration is 0.05% is added, 37 DEG C of digestion 2 ~ 5min, reject Digestive system, use normal saline flushing again 1 ~ 2 time, then the pancreas enzyme-EDTA that mass concentration is 0.25% is added, 3 ~ 6min is digested under 37 DEG C of conditions, add FBS again and stop enzyme reaction, collect Digestive system, by Digestive system at 4 DEG C, centrifugal 5 ~ 15min under the condition of 1500rpm rotation concussion, abandon supernatant, then DMEM/F12 substratum re-suspended cell is added, obtain high purity P0 for amniotic epithelial cells single cell suspension, namely complete.
The beneficial effect of present embodiment has:
(1) can ensure that amnioic epithelium face is smooth after amnion tissue makes amniotic membrane patch, simultaneously amnion back side mucus and reticular tissue and nitrocellulose membrane fit tightly, promote being separated of trysinization efficiency and cell and tissue, short period of time digestion can collect whole amniotic epithelial cells, avoids the reduction of adherence rate.
(2) amniotic epithelial cells is attached to outside amnion surface substrate film, can preferentially and separate tissue, the amnion back side and nitrocellulose membrane are fitted and are hindered the outflow of amnion tissue interstitial cells, in operation, amnion tissue does not need significantly to stir amniotic epithelial cells also can be made fully to be separated with amnion tissue, reduce further the pollution of amnion mesenchymal stem cell.
(3) short period of time cultivates rear use 0.05% pancreatin, 37 DEG C of digestion 2 ~ 5min can make amnion mesenchymal stem cell be separated completely, now amniotic epithelial cells still keeps open and flat adhered state, after purifying, add 0.25% pancreatin, 37 DEG C of digestion 3-6min can obtain high purity amniotic epithelial cells.
Embodiment two: present embodiment and embodiment one unlike: the stripping mode described in step one is blunt separation.Other steps are identical with embodiment one with parameter.
Embodiment three: present embodiment and embodiment one or two are unlike the culture dish of the culture dish A described in step 2, culture dish B and culture dish C to be diameter be 9 ~ 15cm.Other steps are identical with embodiment one or two with parameter.
Embodiment four: one of present embodiment and embodiment one to three unlike: the digestion described in step 2 is at 37 DEG C, under the condition of 60 ~ 120rpm, digestion 15 ~ 20min.Other steps are identical with one of embodiment one to three with parameter.
Embodiment five: one of present embodiment and embodiment one to four unlike: remove in amniotic membrane patch the amniotic epithelial cells of reuniting, being adhered in step 2 with blunt-ended forceps, glass stick or cell curet.Other steps are identical with one of embodiment one to four with parameter.
Embodiment six: one of present embodiment and embodiment one to five unlike: described in step 3 by 1.0 × 10
5/ cm
2inoculum size be inoculated in culturing bottle.Other steps are identical with one of embodiment one to five with parameter.
Embodiment seven: one of present embodiment and embodiment one to six unlike: the final concentration of low sugar DMEM/F12 substratum Concentrations of Epidermal Growth Factor containing Urogastron described in step 3 is 10ng/ml.Other steps are identical with one of embodiment one to six with parameter.
By following verification experimental verification beneficial effect of the present invention:
Test 1, this method tested the separation of a kind of amniotic epithelial cells, cultivate, complete according to the following steps:
One, amnion tissue is separated and sterilization: peeled off on placenta by amnion, then use normal saline flushing 2 ~ 3 times, then soaks 8min in containing the physiological saline of 2% cefoperazone;
Two, amniotic epithelial cells is separated: amnion is cut into 60cm
2bulk, then the epithelial surface of the amnion after cutting is sticked upward on aseptic nitrocellulose membrane, in culture dish A, adds 5mL physiological saline, then the epithelial surface of amnion is posted down in culture dish A, leave standstill 3min, obtain amniotic membrane patch; Amniotic membrane patch is placed in culture dish B, add the pancreas enzyme-EDTA digestion 15min that 20ml mass concentration is 0.25%, then after sealed membrane sealing, culture dish is put into constant-temperature table to digest, obtain Digestive system, add 3ml foetal calf serum again and stop enzyme reaction, then the amniotic epithelial cells of reuniting, being adhered is removed in amniotic membrane patch, amniotic membrane patch being transferred to is equipped with in the culture dish C of physiological saline again, clean adherent amniotic epithelial cells, obtain scavenging solution, collect Digestive system and scavenging solution, through the sieved filter of 100 order cell, obtain amniotic epithelial cells single cell suspension;
Three, amniotic epithelial cells purifying is cultivated: amniotic epithelial cells single cell suspension step 2 obtained is at 4 DEG C, the pelleted by centrifugation 10min of 1500rpm, abandon supernatant, then the low sugar DMEM/F12 substratum re-suspended cell containing Urogastron is added, by 0.9 ~ 1.1 × 10 after mixing
5/ cm
2inoculum size be inoculated in culturing bottle, then be placed in 37 DEG C, CO
2volumetric concentration is cultivate in the incubator of 5%, after cell confluency reaches 85%, reject non-adherent cell, and with normal saline flushing 2 times, then the pancreas enzyme-EDTA digestion that mass concentration is 0.05% is added, 37 DEG C of digestion 3min, reject Digestive system, use normal saline flushing again 1 ~ 2 time, then the pancreas enzyme-EDTA that mass concentration is 0.25% is added, 5min is digested under 37 DEG C of conditions, add FBS again and stop enzyme reaction, collect Digestive system, by Digestive system at 4 DEG C, centrifugal 10min under the condition of 1500rpm rotation concussion, abandon supernatant, then DMEM/F12 substratum re-suspended cell is added, obtain high purity P0 for amniotic epithelial cells single cell suspension, namely complete.
This experimental control tests non-purifying P1 for amniotic epithelial cells, by the amniotic epithelial cells single cell suspension collected at 4 DEG C, the pelleted by centrifugation 10min of 1500rpm, abandon supernatant, then add the low sugar DMEM/F12 substratum re-suspended cell containing Urogastron, by 1.0 × 10 after mixing
5/ cm
2inoculum size be inoculated in culturing bottle, then be placed in 37 DEG C, CO
2volumetric concentration is cultivate in the incubator of 5%, after adherence rate reaches 85%, reject non-adherent cell, and with normal saline flushing 2 times, then the pancreas enzyme-EDTA of 0.25% is added, 3-6min is digested under 37 DEG C of conditions, add FBS again and stop enzyme reaction, collect Digestive system, by Digestive system at 4 DEG C, centrifugal 10min under the condition of 1500rpm, abandon supernatant, then DMEM/F12 substratum re-suspended cell is added, obtain P0 for amniotic epithelial cells single cell suspension, then carry out inoculation and obtain non-purifying P1 for amniotic epithelial cells, namely complete.
For amniotic epithelial cells single cell suspension, Secondary Culture is carried out to the high purity P0 of this test preparation, observes p1 ~ p3 for amniotic epithelial cells with inverted phase contrast microscope, find that P1-P3 all maintains cobblestone-appearance for form, mix without fibroblast-like cells.P1 for amniotic epithelial cells form as shown in Figure 1.From p1 for extracting P1 amniotic epithelial cells for amnion mesenchymal stem cell, with microscopic examination, as shown in Figure 2, amniotic epithelial cells is cobblestone-appearance to result as can be seen from Figure 2, and amnion mesenchymal stem cell is similar to fibroblast-like cells, and morphological differences is obvious.The p1 that control group is cultivated without purifying for amniotic epithelial cells as shown in Figure 3, mixes in amniotic epithelial cells and has fibroblast-like cells, show that the amniotic epithelial cells of not purified culturing process can have amnion mesenchymal stem cell contamination phenomenon.
For amniotic epithelial cells, flow cytometer detection is carried out to the P0 of this test preparation, detected result display is as shown in table 1, as shown in Table 1, in amniotic epithelial cells SSEA-4 expression rate purifying after all significantly improve, and amnion mesenchymal stem cell is expressed without SSEA-4, show to cultivate through purifying the pollution effectively eliminating amnion mesenchymal stem cell.
Table 1 flow cytometer detection result table
| Amniotic epithelial cells after purifying | Non-purifying amniotic epithelial cells | Amnion mesenchymal stem cell | |
| CD29 | 99.66％ | 99.62％ | 99.70％ |
| CD73 | 99.77％ | 99.35％ | 99.52％ |
| CD105 | 84.85％ | 55.24％ | 99.63％ |
| SSEA-4 | 55.66％ | 47.57％ | 0.11％ |
The high purity P0 this test prepared is inoculated for amniotic epithelial cells, obtain the amniotic epithelial cells in p1 generation, the amniotic epithelial cells getting p1 generation carries out Immunofluorescence test, Fig. 4 be under microscope multiple is the condition of 100 p1 for amniotic epithelial cells in Cytokeratin19 immunofluorescence expression figure; Fig. 5 be under microscope multiple is the condition of 100 p1 for amniotic epithelial cells in DAPI immunofluorescence expression figure; Fig. 6 is that p1 expresses integration map for amniotic epithelial cells Cytokeratin19 and DAPI immunofluorescence under microscope multiple is the condition of 100; Fig. 7 be under microscope multiple is the condition of 200 p1 for amniotic epithelial cells in Cytokeratin19 immunofluorescence expression figure; Fig. 8 be under microscope multiple is the condition of 200 p1 for amniotic epithelial cells in DAPI immunofluorescence expression figure; Fig. 9 is that p1 expresses integration map for amniotic epithelial cells Cytokeratin19 and DAPI immunofluorescence under microscope multiple is the condition of 200.From Fig. 4 ~ Fig. 9, P1 for all specific expressed Cytokeratin19 of cell, show to cultivate through purifying to obtain high purity amniotic epithelial cells, pollute without amnion mesenchymal stem cell.
Can ensure that amnioic epithelium face is smooth after the amnion tissue of this test makes amniotic membrane patch, simultaneously amnion back side mucus and reticular tissue and nitrocellulose membrane fit tightly, promote being separated of trysinization efficiency and cell and tissue, short period of time digestion can collect whole amniotic epithelial cells, avoids the reduction of adherence rate; Its deuterzooid test amniotic epithelial cells is attached to outside amnion surface substrate film, can preferentially and separate tissue, the amnion back side and nitrocellulose membrane are fitted and are hindered the outflow of amnion tissue interstitial cells, in operation, amnion tissue does not need significantly to stir amniotic epithelial cells also can be made fully to be separated with amnion tissue, reduce further the pollution of amnion mesenchymal stem cell.Last this test short period of time uses 0.05% pancreatin after cultivating, 37 DEG C of digestion 2-5min can make amnion mesenchymal stem cell be separated completely, and now amniotic epithelial cells still keeps open and flat adhered state, after purifying, add 0.25% pancreatin, 37 DEG C of digestion 3-6min obtain high purity amniotic epithelial cells.
Claims (7)
1. amniotic epithelial cells separation, a method of cultivating, is characterized in that the method completes according to the following steps:
One, amnion tissue is separated and sterilization: peeled off on placenta by amnion, then use normal saline flushing 2 ~ 3 times, then soaks 3 ~ 10min in containing the physiological saline of 1% ~ 3% cefoperazone;
Two, amniotic epithelial cells is separated: amnion is cut into 50 ~ 150cm
2bulk, then the epithelial surface of amnion after cutting is sticked upward on aseptic nitrocellulose membrane, in culture dish A, adds 3 ~ 10mL physiological saline, then the epithelial surface of amnion is posted down in culture dish A, leave standstill 2 ~ 3min, obtain amniotic membrane patch, amniotic membrane patch is placed in culture dish B, add pancreas enzyme-EDTA digestion 15 ~ 20min that 10 ~ 30ml mass concentration is 0.25%, then after sealed membrane sealing, culture dish is put into constant-temperature table to digest, obtain Digestive system, add 2 ~ 5ml foetal calf serum again and stop enzyme reaction, then remove in amniotic membrane patch and reunite, the amniotic epithelial cells be adhered, amniotic membrane patch being transferred to is equipped with in the culture dish C of physiological saline again, clean adherent amniotic epithelial cells, obtain scavenging solution, collect Digestive system and scavenging solution, through the sieved filter of 100 order cell, obtain amniotic epithelial cells single cell suspension,
Three, amniotic epithelial cells purifying is cultivated: amniotic epithelial cells single cell suspension step 2 obtained is at 4 DEG C, the pelleted by centrifugation 5 ~ 15min of 1500rpm, abandon supernatant, then the low sugar DMEM/F12 substratum re-suspended cell containing Urogastron is added, by 0.9 ~ 1.1 × 10 after mixing
5/ cm
2inoculum size be inoculated in culturing bottle, then be placed in 37 DEG C, CO
2volumetric concentration is cultivate in the incubator of 5%, after cell confluency reaches 85%, reject non-adherent cell, and with normal saline flushing 2 times, then the pancreas enzyme-EDTA digestion that mass concentration is 0.05% is added, 37 DEG C of digestion 2 ~ 5min, reject Digestive system, use normal saline flushing again 1 ~ 2 time, then the pancreas enzyme-EDTA that mass concentration is 0.25% is added, 3 ~ 6min is digested under 37 DEG C of conditions, add FBS again and stop enzyme reaction, collect Digestive system, by Digestive system at 4 DEG C, centrifugal 5 ~ 15min under the condition of 1500rpm rotation concussion, abandon supernatant, then DMEM/F12 substratum re-suspended cell is added, obtain high purity P0 for amniotic epithelial cells single cell suspension, namely complete.
2. a kind of amniotic epithelial cells separation according to claim 1, the method for cultivating, is characterized in that the stripping mode described in step one is blunt separation.
3. a kind of amniotic epithelial cells according to claim 1 method that is separated, cultivates, is characterized in that the culture dish A described in step 2, culture dish B and culture dish C to be diameter is the culture dish of 9 ~ 15cm.
4. a kind of amniotic epithelial cells separation according to claim 1, the method for cultivating, is characterized in that the digestion described in step 2 is at 37 DEG C, under the condition of 60 ~ 120rpm, and digestion 15 ~ 20min.
5. a kind of amniotic epithelial cells separation according to claim 1, the method for cultivating, is characterized in that removing in amniotic membrane patch the amniotic epithelial cells of reuniting, being adhered in step 2 with blunt-ended forceps, glass stick or cell curet.
6. a kind of amniotic epithelial cells according to claim 1 method that is separated, cultivates, it is characterized in that described in step 3 by 1.0 × 10
5/ cm
2inoculum size be inoculated in culturing bottle.
7. a kind of amniotic epithelial cells separation according to claim 1, the method for cultivating, is characterized in that the final concentration of the low sugar DMEM/F12 substratum Concentrations of Epidermal Growth Factor containing Urogastron described in step 3 is 10ng/ml.
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| CN109528772A (en) * | 2017-09-22 | 2019-03-29 | 中国福利会国际和平妇幼保健院 | Application of the human amnion membrane secretion factor in the drug that ovarian function is repaired in preparation |
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| CN106520676A (en) * | 2016-12-09 | 2017-03-22 | 博雅干细胞科技有限公司 | Method for preparing human amniotic membrane epithelial cells from human placenta amnion and application thereof |
| CN106520676B (en) * | 2016-12-09 | 2019-07-26 | 博雅干细胞科技有限公司 | The method and its application of human amnion membrane are prepared from Human plactnta amnion |
| CN106834218A (en) * | 2017-01-06 | 2017-06-13 | 庞希宁 | People's amnioic epithelium stem cell serum-free culture medium and its cultural method |
| CN106834217A (en) * | 2017-04-18 | 2017-06-13 | 遵义医学院附属医院 | A kind of method for promoting human amnion membrane amplification in vitro and application |
| CN109528772A (en) * | 2017-09-22 | 2019-03-29 | 中国福利会国际和平妇幼保健院 | Application of the human amnion membrane secretion factor in the drug that ovarian function is repaired in preparation |
| CN109182260A (en) * | 2018-09-11 | 2019-01-11 | 邵勇 | A kind of method of in vitro culture fetal membrane mescenchymal stem cell |
| CN113015789A (en) * | 2018-11-08 | 2021-06-22 | 整合培育株式会社 | Animal cell growth promoter, medium for animal cell culture, and animal cell culture device |
| CN114480251A (en) * | 2021-12-31 | 2022-05-13 | 浙江金时代生物技术有限公司 | Amniotic epithelial cell culture method |
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